The present invention relates to a process for detecting threonine or serine (threonine/serine) kinase activity in an immunoassay. The invention further relates to a kit for carrying out the assay and to a preferably luminescently labelled ligand.
Protein kinases usually catalyse the transfer of the γ-phosphate group from ATP to a serine, threonine or tyrosine residue of an acceptor protein. These enzymes play an important role in signal transduction within cells. Detecting the activity of protein kinases would be useful for the high-throughput screening of chemical libraries. Inhibitors or activators of kinase activity, particularly low-molecular weight compounds, could eventually be developed into drugs used for the treatment of e.g. ischaemic heart disease, liver failure, diabetic neuropathies, stroke, neurodegenerative disorders including Parkinsons disease and Alzheimers disease, inflammatory diseases including asthma, rheumatoid arthritis, inflammatory bowel disease, septic shock and cancer.
It is known that mitogen-activated protein kinases (MAPK) (also referred to as stress activated protein kinases, SAPK) mediate many of the cellular effects of growth factors, cytokines and stress, leading to cell growth, differentiation and oncogenesis. MAPK/SAPK activation requires dual phosphorylation on threonine and tyrosine within the motif threonine-Xaa-tyrosine, where Xaa represents proline in the c-Jun NH2-terminal kinases (JNKs). This activation is catalysed by several MAPK-kinases (e.g. MKK4(SKK1) or MKK7 (SKK4)) which phosphorylate both the threonine and tyrosine residues of the threonine(183)-proline-tyrosine(185) motif within the active site loop of JNK1/2/3. The MKK7 kinase has a high preference for the threonine residue whereas MKK4 preferentially phosphorylates tyrosine. This synergistic activation of JNK1/2/3 is described by Lawler S. et al in Current Biology 8, 1387 to 1390 (1998) and Fleming Y et al. (Biochemical Journal, 352, 145–154, 2000). Up to now the identification of an appropriate substrate which could be used to screen for specific MKK7 inhibitors is complicated by the fact that there are no synthetic substrate peptides described in the literature. The situation becomes even more critical in homogeneous assays as a potential MKK7 substrate peptide should have both a reasonable Km for MKK7 and a high affinity for the phospho-threonine specific detection antibody in parallel. Although generic anti-phospho-tyrosine antibodies with high affinity and specificity are available for tyrosine-directed kinases (e.g. p60c-src kinase), attempts to develop generic anti-phospho-threonine/anti-phospho-serine antibodies for the same purpose have been to date less successful.
Antibodies which bind to phosphorylated threonine or serine residues with high affinity only recognize the phosphorylated amino acid in the context of the surrounding peptide sequence. For antibodies binding to JNK1/2/3 or peptide derivatives thereof, recognition is therefore dependent on two criteria: 1) MKK7-dependent phosphorylation of threonine and 2) phosphorylation of the tyrosine amino acid residue. If one of the two criteria is not fulfilled, an antibody raised against fully activated JNK1/2/3 will not specifically detect the phosphorylation site.
In Anal. Biochem. 255, 257 to 262 (1998) a fluorescence polarization (FP) competition immunoassay for tyrosine kinases using an appropriate substrate for this kinase is described. The kinase reaction was performed by incubation of the peptide substrate with ATP and lymphoid T-cell protein tyrosine kinase followed by termination of the reaction with EDTA plus a fluorescein-phosphopeptide. Following the addition of an anti-phosphotyrosine antibody, the fluorescence polarization signal was measured. The phosphorylated product formed in the assay competes with the fluorescein-phosphopeptide for binding to the anti-phosphotyrosine antibody. The kinase activity results in a reduction of the FP signal and the FP signal is therefore inversely proportional to the phosphorylated product formed in the reaction.
However, such an approach for a generic assay principle for serine/threonine kinases is not realizable to date. This is because there are no anti-phospho-serine/-threonine antibodies available that bind specifically, and with high affinity, to phosphoserine or phosphothreonine residues in the absence of additional specific amino acid sequences adjacent to the phosphorylated amino acid.